The heterotrimeric G protein plays an important regulatory role in the human pathogenic fungus Cryptococcus neoformans. The G1 subunit Gpa1 governs a conserved cAMP-dependent signaling pathway that regulates virulence, but the necessary components of a Gpa1 heterotrimeric G protein complex are not known. Using Gpa1 as protein bait and through a yeast two-hybrid screen, we have identified a novel G2- like/RACK1 protein, Gib2, which exhibits a G2-like function by association with Gpa1 and G3 subunits Gpg1 and Gpg2. In addition, we found that Gib2 regulates formation of virulence factors melanin and capsule through modulation of intracellular cAMP in a gpa1 mutant strain, and Gib2 exhibits shared and distinct functions across serotypes: essential in var. neoformans, but required for full virulence of var. grubii. Four specific aims are proposed to characterize the mechanism by which Gib2 functions as an atypical G2 in regulation of cAMP, as a virulence factor, and as a multi-functional protein. Identification and characterization of Gib2-mediated atypical G protein signaling and Gib2 function is significant not only for our understanding of signal transduction mechanisms regulating cellular growth, differentiation, and pathogenesis but also in our pursuing of novel targets for antifungal therapy. Relevance: Cryptococcus neoformans is a prevalent opportunistic pathogen that causes fungal meningoencephalitis in humans. G protein-mediated signal transduction plays an important regulatory role in the physiology and pathogenesis of the fungus. Understanding the molecular events that underlie G protein signaling may help to reveal novel targets for antifungal therapy.